1. Field of the Invention
The invention is generally directed to alginases. In particular, the present invention is directed to alginases found in Microbulbifer degradans and systems containing such alginases and methods of cloning, purifying and/or utilizing such alginases.
2. Background of the Invention
Saccharophagus degradans strain 2-40 (herein referred to as “S. degradans 2-40” or “2-40”) is a representative of an emerging group of marine bacteria that degrade complex polysaccharides (CP). S. degradans has been deposited at the American Type Culture Collection and bears accession number ATCC 43961. S. degradans 2-40, formerly known and referred to synonomously herein as Microbulbifer degradans strain 2-40 (“M. degradans 2-40”), is a marine γ-proteobacterium that was isolated from decaying Sparina alterniflora, a salt marsh cord grass in the Chesapeake Bay watershed. Consistent with its isolation from decaying plant matter, S. degradans strain 2-40 is able to degrade many complex polysaccharides, including cellulose, pectin, xylan, and chitin, which are common components of the cell walls of higher plants. S. degradans strain 2-40 is also able to depolymerize algal cell wall components, such as agar, agarose, and laminarin, as well as protein, starch, pullulan, and alginic acid. In addition to degrading this plethora of polymers, S. degradans strain 2-40 can utilize each of the polysaccharides as the sole carbon source. Therefore, S. degradans strain 2-40 is not only an excellent model of microbial degradation of insoluble complex polysaccharides (ICPs) but can also be used as a paradigm for complete metabolism of these ICPs. ICPs are polymerized saccharides that are used for form and structure in animals and plants. They are insoluble in water and therefore are difficult to break down.
Microbulbifer degradans strain 2-40 requires at least 1% sea salts for growth and will tolerate salt concentrations as high as 10%. It is a highly pleomorphic, Gram-negative bacterium that is aerobic, generally rod-shaped, and motile by means of a single polar flagellum. Previous work has determined that 2-40 can degrade at least 10 different carbohydrate polymers (CP), including agar, chitin, alginic acid, carboxymethylcellulose (CMC), β-glucan, laminarin, pectin, pullulan, starch and xylan (Ensor, Stotz et al. 1999). In addition, it has been shown to synthesize a true tyrosinase (Kelley, Coyne et al. 1990). 16S rDNA analysis shows that 2-40 is a member of the gamma-subclass of the phylum Proteobacteria, related to Microbulbifer hydrolyticus (Gonzalez and Weiner 2000) and to Teridinibacter sp., (Distel, Morrill et al. 2002) cellulolytic nitrogen-fixing bacteria that are symbionts of shipworms.
These exo- and extra-cellular structures (ES) include small protuberances, larger bleb-like structures that appear to be released from the cell, fine fimbrae or pili, and a network of fibril-like appendages which may be tubules of some kind. Immunoelectron microscopy has shown that agarases, alginases and/or chitinases are localized in at least some types of 2-40 ES. The surface topology and pattern of immunolocalization of 2-40 enzymes to surface protuberances are very similar to what is seen with cellulolytic members of the genus Clostridium. 
2-40 is a gram negative, pleomorphic, motile with a means of a single polar flagellum (see FIG. 1). Cells average 0.5 μm in width and 1.5-3.0 μm in length (Andrykovich and Marx 1988). During late logarithmic-stationary phases of growth, a black pigment, identified as true melanin (Stosz 1994), is produced and cells become elongated (Marx 1986).
The G+C content of 2-40 is 45.66%, as determined by the ATCC (Stosz 1994). 2-40 is catalase- and peroxidase-positive (González and Weiner 2000). It is a strict aerobe capable of respiratory, but not fermentative, metabolism, and requires both sea salts and carbohydrates for growth (Marx 1986, Stosz 1994). It does not form spores or accumulate β-hydroxybutyrate. As an estuarine bacterium, 2-40 is capable of reproduction in a wide range of temperatures (5° C. to 40° C.) and can tolerate 2-10% sea salt. 2-40 can also grow in pH range of 4.5-10, with optimum pH of 7.5 (Gonzalez and Weiner 2000).
2-40 is unique in its capability to degrade numerous insoluble complex polysaccharides (ICP) including alginic acid, agar, cellulose, chitin, glucan, pectin, pullulan, starch and xylan (Whitehead 1997). In addition to its ability to degrade these carbohydrates, 2-40 is also capable of producing lipases, proteases, and tyrosinase (Marx 1986, and Stosz 1994).
Previous studies showed that the cell surface morphology of 2-40 changed when the organism was grown on different insoluble complex polysaccharides (Whitehead 1997). When 2-40 was cultivated on either chitin or agarose, scanning and transmission electron microscopy revealed that the presence of cell surface protuberances, hydrolysomes, correlated to the degradation of these two complex polysaccharides. Other changes in the cell topology and morphology were detected during late growth stages. These changes included production of membranous tubules containing agarases and chitinases. These morphological changes may correlate with the ability of 2-40 to survive dynamic changes in the estuarine ecosystem (Chakravorty 1998 and Whitehead 1997).
Preliminary studies suggested that 2-40 be assigned to genus Alteromonas (Andrykovich and Marx 1988). However, a recent search in the MIDI database revealed that Marinobacterium georgiense is the closest relative to 2-40 based on fatty acid profile. It also revealed a comparatively low level of similarity with that of Microbulbifer hydrolyticus IRE 31, the closest strain to 240 according to its 16S rDNA (Gonzalez and Weiner 2000). Moreover, the GenBank search showed that 2-40 has 93% similarity with Microbulbifer hydrolyticus, and 91.2% similarity with a cellulytic nitrogen-fixing bacterium, isolated from the gland of Deshayes in three different species of shipworm (González and Weiner 2000). While the taxonomy is not yet fully settled, based upon the 16S rDNA analysis, strain 2-40 was placed in genus Microbulbifer as a new species, Microbulbifer degradans. It is a member of the γ-subclass of the phylum Proteobacteria (González and Weiner 2000 and Weiner et al., 2000).
Marsh grass Spartina alterniflora is found to be the most common species in the salt marshes of the east coast of North America (Ford 1993). In addition to S. alterniflora, which is a dominant species at mid level of elevations, Spartina patens and Distichlis spicata dominate at high elevation while Zostera marina and brown algae are common in low marsh elevation (Chakravorty 1998). Salt marsh grass supports a wide range of algal population, including green, brown, blue-green, and red algae, in addition to a diverse bacterial, fungal, protozoan, and invertebrate populations (Stosz 1994). 2-40 was shown to have capabilities to produce different degradative enzyme systems and to utilize a variety of substrates, all of which increase the organism's ability to survive in this environment. It can also naturally recycle several ICPs, thus may be employed in bioremediation (Chakravorty 1998).
In natural environments, numerous amounts and various kinds of ICPs are formed and accumulate leading to the requirement for efficient mechanisms for their degradation. As part of the carbon cycle, they are recycled to their primary elements (Whitehead 1997 and Chakravorty 1998). These ICP, composed of homo- and heteropolysaccharides, account for substantial agriculture, aquaculture and algalculture wastes. With the exception of starch, these compounds compose the cell wall structure in plants and fungi (Whitehead 1997). Because of their binding, branching sugar composition, and complexed formation with other polymers, the degradation of ICPs is not a trivial process. However, these ICPs can be hydrolyzed by microorganisms to produce monosaccharide feedstock. For example, in the marine environment, around 1011 tons of chitin wastes are produced annually, yet, apart from living or recently living biota, only traces of it are found in marine sediments. This is explained by the presence of microorganisms that degrade chitin and recycle the carbon and nitrogen (Salyers et al., 1996). For economical and environmental considerations, biomerediation, using prokaryotes is an efficient way to recycle ICP. Bacteria and fungi degrade ICP to provide saccharide feedstock (Salyers et al., 1996).
In addition to feedstock, degradation of alginic acid yields 4-deoxy-L-erythro-hex-4-ene-pyranosylurinate containing oligosaccharides, which are thought to be active biological molecules. These oligosaccharides can elicit plant germination, shoot elongation and root growth promoting activities (Natsume et al., 1994). They also stimulate the growth of Bifidobacteria, a useful food industry organism (Akiyama et al., 1992).
Alginic acid is a high molecular weight linear polysaccharide polymer produced mainly by seaweed, as well as many species of marine algae and certain bacteria (Linhardt et al., 1986 and Chakravorty 1998). It is comprised of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G), (See FIG. 2), (Gacesa 1992). The salts of alginic acid are commonly referred to as alginate, which include: ammonium alginate, calcium alginate, potassium alginate, and sodium alginate (Chakravorty 1998). The primary structure of alginic acid is highly variable based on the monomer ratios and distribution of monomers into homopolymeric blocks (GG or MM) or heteropolymeric blocks (MG) (Doubet and Quatrano 1982).
The composition of alginate greatly depends on the producing organism and its physiology. Algal alginates are unbranched (1-4) linked glycuronans containing residues of β-D-mannosyluronic acid and the C5 epimer α-L-gulosyluronic acid, whereas bacteria normally produce their alginate being invariably O-acetylated, where O-acetyl groups are found on the 2 and/or 3 positions of D-mannuronate residues (Gacesa 1988). This acetylation often affects water-binding properties and ion-binding selectivity of the polymer (Wong et al., 2000). The level of alginate's susceptibility to degradation is normally influenced by both the block structure and degree of O-acetylation within the macromolecule (Wong et al., 2000).
Algal and bacterial alginates also differ in their molecular weight. Alginate produced by A. vinelandii has a molecular weight of 2×106 Da, whereas algal alginates have a range of 48000 to 186000 Da; in general, bacterial alginates usually have higher molecular mass than algal polymer (Pe{umlaut over (n)}a et al., 2002).
Alginate, a viscous polysaccharide, is found in the cell wall of the brown seaweeds (Phaeophyceae) and produced by several species of bacteria. Like its structure, the function of alginate varies depending on its source. In brown algae, alginate comprises about 60% of the cell wall mass of Fucus distichus (Doubet and Quatrano 1982). Approximately 22,000 tons/annum of alginate are extracted from numerous brown algal genera to be used in a variety of applications by the food, pharmaceutical and other industries. Most of the commercial alginate is extracted mainly from genera Macrocystis, Laminaria, and Ascophyllum (Wong et al., 2000). The brown algae alginate is believed to function as an intercellular skeletal matrix (Ertesvåg et al., 1995).
In addition to the brown algae, alginate is also produced by two bacterial families, Azotobacteriaceae and Pseudomodacease (Wong et al., 2000). Azotobacter vinelandii, A. chroococcum, Pseudomonas aeruginosa and other Pseudomonads synthesize alginate as an extracellular polysaccharide (Gacesa 1992), and as a major component of many biofilms (Weiner et al., 1998). It has its ability to form viscous solutions at relatively low concentrations and to form gels with Ca+2 (Davidson et al., 1976).
Alginate is enzymatically degraded by a group of enzymes that catalyze the β-elimination of the 4-O— linked glycosidic bond forming unsaturated uronic acid-containing oligosaccharides (Preiss and Ashwell 1962a, Kiss 1974, Caswell et al., 1986, Gacesa 1992, and Wong et al., 2000).
Alginases, typically lyases, are members of the class polysaccharide lyases, or eliminases, (EC 4.2.2.-). They normally act in a wide range of naturally acidic polysaccharides. Enzymes of this class have low or intermediate molecular weight (20-110 kDa) and are characteristically, usually, monomeric, having the same molecular weight when determined under reducing or non-reducing conditions. They act through a β-elimination mechanism (Haugen et al., 1990), rather than hydrolysis, to cleave certain glycosidic linkage in the acidic polysaccharides. This reaction results in unsaturated oligosaccharide products (uronic acid residues) at the new non-reducing end (Haugen et al., 1990, Linhardt et al., 1986). Polysaccharides cleaved by elimination generally contain a carboxylate group on the carbon adjacent to the glycosidic linkage (Haugen et al., 1990, Gacesa 1992). However, one group reported that alginases are hydrolyases (Schaumann and Weide, 1990), where the enzyme was isolated from marine fungi, Dendryphiella salina and Asteromyces cruciatus. The mechanism of action of this enzyme, though, is not fully understood (Gacesa 1992).
Alginate lyase (EC 4.2.2.3) catalyzes the reaction of alginate degradation by elimination mechanism (Romeo and Preston 1986b, Linhardt et al, 1986, Gacesa 1992, Wong et al., 2000). This reaction targets the glycosidic 1→4 O-linkage between alginate monomers. The results are: a) formation of double bond between the C4 and C5 of the six-carbon ring, from which the 4-O-glycosidic bond is eliminated; b) depolymerization of alginate; and finally c) a product containing 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid as the non-reducing terminal (Gacesa 1992 and Wong et al., 2000).
Alginases are normally utilized to degrade alginate as a carbon source; however, interestingly alginate-producing organisms are not usually capable of growing on alginate as the sole source of carbon. On the other hand, organisms capable of utilizing alginic acid as a sole source of carbon produce both exo- and endolytic alginases, unless they exhibit commensalisms with another organisms to degrade the complex polysaccharide to monomeric subunits (Gacesa 1992).
Alginate lyases have been isolated from different organisms including marine algae, marine bacteria, marine mollusk, fungi, and a wide variety of microorganisms (Hansen et al., 1984, Gacesa 1992, Wong et al., 2000).
TABLE 1.1Alginase producing organisms.OrganismSourceEnzymeReference2-40 (Microbulbifursalt marsh grassalginic acid lyaseMarx 1986, Stosz 1994,degradans)bacterial isolatesWhitehead 1997, thisstudyAgrobacterium tumefaciensGenomealginic acid lyaseGoodne et al., 2001str. C58sequence1Alginovibrio aqualiticusmarine bacteriumalginic acid lyaseStevens & Levin 1977Alteromonas sr. strain KLIAmarine bacteriumalginic acid lyaseSawabe et al., 1997Alteromonas spp.soil bacteriumalginic acid lyaseVilter 1986Aplysia spp.Mollusksalginic acid lyaseKloareg et al., 1989Asteromyces cruciatusmarine bacteriumalginic acidSchaumann & Weidehydrolyase1990Azotobacter chroococcumsoil bacteriumalginic acid lyaseKennedy et al., 1992Azotobacter vinelandiisoil bacteriumalginic acid lyaseKennedy et al., 1992Bacillus circulanssoil bacteriumalginic acid lyaseHansen et al., 1984Bacillus haloduransGenomealginic acid lyaseTakami et al., 1999sequence1Bacteriophage that infectsPhagealginic acid lyaseDavidson et al., 1977A. vinelandiiBeneckea pelagiamarine bacteriumalginic acid lyasePitt & Raisbeck 1978Chlorella virusvirusalginic acid lyaseSuda et al., 1999Choromylitis meridonalisMolluskalginic acid lyaseSeiderer et al., 1982Clostridium grantiisoil bacteriumalginic acid lyaseMountfort et al., 1994Corynebacterium spp.soil/marinealginic acid lyaseMatsubara et al., 1998bacteriumDollabella auricularMolluskalginic acid lyaseNishizawa et al., 1968Enterobacter cloacaesoil/marinealginic acid lyaseShimokawa et al., 1997bacteriumFucus zygotesbrown algaealginic acid lyaseVreeland & Laetsch1990Haliotis corrugateMolluskalginic acid lyaseLinhardt et al., 1986Haliotis rufescensMolluskalginic acid lyaseLinhardt et al., 1986Haliotis tuberculataMolluskalginic acid lyaseKloareg & Quatrano 1987Katherina tunicateMolluskalginic acid lyaseKloareg & Quattrano 1987Klebsiella pneumoniaesoil/marinealginic acid lyaseBoyd &Turvey 1977,bacteriumLange et al., 1989Laminaria digitatabrown algaealginic acid lyaseMadgwick et al., 1978Littorina spp.brown algaealginic acid lyaseElaykova & Favorov 1974Mesorhizobium lotiGenomealginic acid lyaseKaneko et al., 2000sequence1Pelvetia canalitulatabrown algaealginic acid lyaseMadgwick et al., 1978Perna pernaMolluskalginic acid lyaseSeiderer et al., 1982Photobacterium spp.marine bacteriumalginic acid lyaseRomeo & Preston 1986aPseudoalteromonasalginic acid lyaseSawabe et al., 2001elyakoviiPseudomonas alginovoramarine bacteriumalginic acid lyaseChavagnat et al., 1996Pseudomonas aeruginosamarine bacteriumalginic acid lyaseLinker et al., 1984Pseudomonas maltophiliamarine bacteriumalginic acid lyaseSutherland & Keen 1981Pseudomonas putidamarine bacteriumalginic acid lyaseConti et al., 1994Pseudomonas syringae pv.Plant pathogenalginic acid lyaseOtt et al., 2001phaseolicolaPseudomonas syringae pv.Plant pathogenalginic acid lyasePreston et a., 2000syringaeSalmonella enterica subsp.Genomealginic acid lyaseParkhill et al., 2001enterica serovar typhisequence1Sphingomonas species Alalginic acid lyaseYoon et al., 2000Spinula solidissimaMolluskalginic acid lyaseJacober et al., 1980Staphylococcus aureusGenomealginic acid lyaseBaba, et al., 2002subsp. aureus MW2sequence1Turbo corrutusMolluskalginic acid lyaseMuramatsu et al., 1977Undaria pinnatifidabrown algaealginic acid lyaseWatanabe & Nishizawa1982Vibrio alginolyticusmarine bacteriumalginic acid lyaseKitamikado et al., 1992Vibrio harveyimarine bacteriumalginic acid lyaseKitamikado et al., 1992Xanthomonas axonopodisGenomealginic acid lyaseDa Silva et al., 2002pv. citri str. 306sequence1Yersinia pestis KIMGenomealginic acid lyaseDeng, et al., 2002sequence11Genome sequence obtained from National Center for Biotechnology Information (NCBI) data base, (www.ncbi.nlm.nih.gov). Table partially adapted from Chakravorty 1998.
Table 1.2 illustrates alginase properties from some marine and other gram-negative bacteria. The table shows that optimum pH for most alginases ranges around neutral; specifically it falls in between 6-8.5, while optimum temperature for alginases from different sources has a broad range.
Alginate monomers are linked by 4-O-glycosidic bonds. These bonds can be chemically degraded either by lyase activity (Haug et al., 1967, Doubet and Quatrano 1982) or reportedly by alkali-catalyzed β-elimination (Kiss 1974). The alkali may actually disrupt all polysaccharide linkages being non specific for alginate linkages.
Alginase, and well as other degradative enzymes produced by 2-40 could be useful bioremediation tools. As human population increases and more food is required, agricultural, aquacultural, and algalcultural wastes also increase and can become a serious problem. The wastes are mostly recalcitrant complex carbohydrates, namely cellulose, chitin and agar. The complex carbohydrates from natural and human practices are composed of monosaccharide, many of which can provide valuable feedstock when hydrolyzed. The degradative protuberances of 2-40 could be used as bioremediation tools when used as concentrated, organized, protective enzyme packets.
TABLE 1.2Alginate lyases from marine and gram-negative bacteria: localization and properties.MolecularEndo/weightOptSourceLocalizationaExolytic(kDa)plpHOpt TRef.Marine bacteriaAlginovibrio aquatilisExtracellularEndolytic110—8—Stevens andLevin 1977.Alteromonas sp.ExtracellularEndolytic324.77.530Sawabe et al.,Strain H-41992, Sawabe etal., 1997.Beneckae pelagiaIntracellular———825Sutherland andKeen 1981Extracellular—Pitt and Raisbeck1978Halomonas marinaIntracellular—397.78——Kraiwattanapon etal., 1999Photobacterium spPeriplasm (R)Endolytic306——Malissard et al.,(ATCC 433367)1995. Malissard etal., 1993.Pseudomonas sp.Intracellular/Endolytic94—7.5—Muramatsu and(marine)Extracellular32—7.5—Sogi, 1990PseudomonasExtracellularEndolytic325.57.5—Boyen et al., 1990alginovora(strain X017)Vibrio sp. (marineExtracellular———8.545Chavagnat F, etbacterium)al. 1996. Takeshitaand Muramatsu1995 Takeshita etal., 1993Vibrio alginolyticusExtracellularEndolytic474.68.2—Kitamikado et al.,ATCC 177491992, Kitamikadoet al., 1990.Vibrio halioticoliNANA252aaNANANAWong et al., 2000.Vibrio harveyi AL-128ExtracellularEndolytic574.37.8—Kitamikado 1992,Kitamikado et al.,1990, Tseng et al.,1992Gram-negative bacterialA. chroococcumPeriplasmEndolytic43——30Peciña andPaneque 1994,Peciña et al., 1999A. vinelandiiIntracellular—~50—7.5—Davidson et al.,1977Enterobacter cloacaeExtracellularEndolytic32-388.97.830Nibu et al., 1995M-1pl: isoelectric point, the pH at which a molecule carriers no net electric charge.Opt pH: optimum pH,Opt T: Optimum temperature.
TABLE 1.2Alginate lyases from marine and gram-negative bacteria: localization and properties (cont'd).MolecularEndo/weightOptSourceLocalizationaExolytic(kDa)plpHOpt TRef.K. aerogenes typeIntracellularEndolytic28-31.6—737Lange et al., 1989.25ExtracellularEndolytic——7—Boyd and Turvey1977,Haugen et al., 1990K. pneumoniaeExtra/intracellular—288.9——Caswell et al., 1989subsp. aerogenes(R)P. syringae pv.PeriplasmicEndolytic408.2742Wong et al., 2000syringaePseudomonas sp.Intracellular—90, 72,———Kraiwattanapong etOS-ALG-960, 54al., 1997Sphingomonas sp.CytoplasmEndolytic60 9.037.5-8.570Yonemoto et al.,1991, Yonemoto etal., 1993, Yonemotoet al., 1992.StreptomycesNANA259aaNANANARedenbach et al.,coelicolor1996—: Not determined,NA: not available,aLocalization in native culture, except R,R = recombinant expression in E. coli.N/A: not available,Opt: optimum,T: temperature ° C.,aa: amino acid residues.Adapted from Wong et al., 2000.
Alginase has potential medical importance. The alginate glycocalyx abundantly produced by mucoid strains of Pseudomonas aeruginosa is considered a major virulence factor in endocarditis (Bayer et. al., 1992). It also contributes to the morbidity and mortality associated with pseudomonal infections in patients with cystic fibrosis (Dinwiddie 1990, Gacesa, P. 1988) where alginate promotes attachment to the host cell and inhibits the phagocytosis (Bayer et al., 1992, Gacesa 1992, Monday and Schiller 1996).
Pseudomonas aeruginosa is one of the most important opportunistic human pathogens, causing septicemia and severe or even lethal infection to the respiratory tract, urinary tract, intestines and many other sites (Cross et al., 1983). This organism exhibits inherent resistance to a wide range of antibiotics, which makes infection with this pathogen common and hard to treat (Monday and Schiller 1996).
In a recent study, the effect of alginase on the polymorphonuclear leukocyte (PMN)-directed and antibiotic-mediated phagocytosis and killing of mucoid P. aeruginosa was investigated both in vitro and in vivo. The study showed that pretreating of mucoid P. aeruginosa strain 144MR with alginase significantly enhanced PMN phagocytosis, rendering the bacteria more susceptible to PMN-mediated killing than 144MR cells not treated with alginase (P<0.05), approximating the levels of that of non-mucoid strain, 144NM. More importantly, the study also showed that treating the mucoid strain 144MR with alginase caused a significant removal of bacterial cell surface alginate as determined by immunofluroscence staining with a murine monoclonal anti-alginate antibody (Bayer et. al., 1992).